Glucocorticoids attached to nitrate esters via an aromatic linker in position 21 and their use in ophthalmology

ABSTRACT

The invention relates to nitrooxy derivatives of fluocinolone acetonide, triamcinolone acetonide, betamethasone and beclomethasone, methods for their preparation, pharmaceutical compositions containing these compounds, and methods of using these compounds and compositions for treating diabetic macular edema, diabetic retinopathy, macular degeneration, age-related macular degeneration and other diseases of retina and macula lutea.

This application is a National Stage entry of International ApplicationNo. PCT/EP2009/058572, filed Jul. 7, 2009, which claims the benefitunder 35 U.S.C. §119(e) of U.S. Provisional Patent Application No.61/122,896 filed Dec. 16, 2008, now pending, which claims the benefit ofU.S. Provisional Patent Application No. 61/085,294 filed Jul. 31, 2008,now abandoned, the disclosure of the prior application is incorporatedin its entirety by reference.

The invention relates to nitrooxy derivatives of steroids, methods fortheir preparation, pharmaceutical compositions containing thesecompounds, and methods of using these compounds and compositions fortreating ocular diseases, in particular diabetic macular edema, diabeticretinopathy, macular degeneration, age-related macular degeneration andother diseases of retina and macula lutea.

The retina is the part of the eye that is sensitive to light. The maculalutea is the region of the retina that allows us to read and recognizefaces. Diseases of the macula, such as age-related macular degenerationand diabetic macular edema, account for major causes of blindness.

To combat these diseases, a variety of drugs have been investigated fortheir effects on blinding disorders.

Currently, these drugs are delivered to the macula and retina viasurgical procedures such as intravitreal or periorbital injections, orvia systemic routes. Surgical methods often require repeated injectionsand may lead to serious ocular complications, including endophthalmitis,retinal detachment, and vitreous hemorrhage. Likewise, systemicadministration is associated with a variety of potential systemic sideeffects and with the difficulty of delivering therapeutic levels of thedrugs to the retina.

Recently, there have been many reports of the effectiveness ofintravitreal triamcinolone acetonide for the treatment of diffusemacular edema, refractory to laser treatment.

Intravitreal triamcinolone injections are however associated with manyocular complications. The complications of intravitreal triamcinolonetherapy include steroid induced elevation of intraocular pressure,cataractogenesis, post-operative infectious and non-infectiousendophthalmitis, and pseudo-endophthalmitis.

At present chemotherapy, steroids and carbonic anhydrase inhibitors asmajor efficacy are used in symptomatic therapy, but their effectivenessis not established and their administration for a long time leads tooccurrence of side effects such as cataract, steroid induced elevationof intraocular pressure, glaucoma, and infections thus continuous use ofthese drugs in chronic diseases, such as diabetes mellitus, is difficultunder the circumstances.

EP 0929565 discloses compounds of general formula B—X₁—NO₂ wherein Bcontains a steroid residue, in particular hydrocortisone, and X₁ is abivalent connecting bridge which is a benzyl ring, an alkyl chain or aether. The compounds may be used in the treatment of ocular disorders.

EP 1 475 386 discloses compounds of formula A-B—C—NO₂ wherein A containsa steroid residue and B—C is a bivalent connecting bridge which containsan antioxidant residue. The compounds may be used in the treatment ofoxidative stress and/or endothelial dysfunctions.

In the disclosed compounds the antioxidant linker is linked to the 21-OHof the steroid through a carboxylic group forming an ester bond.

WO 03/64443 discloses compounds of general formula B—X₁—NO₂ wherein Bcontains a steroid residue and X₁ is a bivalent connecting bridge whichis a benzyl ring or a heterocyclic linker. The compounds may be used inthe treatment of ocular diseases

WO 07/025632 discloses compounds of formula R—Z—X—ONO₂ wherein R—Xcontains triamcinolone acetonide, betamethasone valerate or prednisoloneethylcarbonate residue and X₁ is a bivalent connecting bridge which isan aromatic ring, an alkyl chain, an ether, ferulic acid, vanillic acidor an heterocyclic ring. The compounds may be used in the treatment ofskin or mucosal membrane diseases and in particular in the treatment ofatopic dermatitis, contact dermatitis and psoriasis.

F. Galassi et al. Br J Ophthalmology 2006, 90, 1414-1419 discloses theeffects of an dexamethasone 21-[(4-nitrooxymethyl)]benzoate in a modelof experimental corticosteroid-induced glaucoma in the rabbit. TheNO-releasing dexamethasone was administered topically twice a day, theresults show that the compound may prevent the intraocular pressureincrease, the impairment of retro bulbar circulation, and themorphological changes in the ciliary bodies possibly induced by topicaltreatment with corticosteroids.

It is an object of the present invention to provide nitrooxy-derivativesof steroids for treating inflammatory diseases.

Another object of the present invention to provide nitrooxy-derivativesof steroids for the prevention or the treatment of ocular diseases, inparticular diabetic macular degeneration, diabetic retinopathy,age-related macular degeneration and other diseases of retina and maculalutea. In one aspect of the invention, one or more of these compoundsreduce the side effects associated with the standard therapy withsteroids. In a further embodiment, one or more of these compoundspossess improved pharmacological activity compared to current standardtherapy.

An object of the present invention is a compound of formula (I) or asalt or a stereoisomer thereof

wherein

R₁ is OH, R₂ is CH₃, or R₁ and R₂ taken together are the group offormula (II)

R₃ is a hydrogen atom or F and R₄ is F or Cl,

with the proviso that:

-   -   when R₁ is OH and R₂ is CH₃, R₃ is a hydrogen atom;    -   when R₁ and R₂ taken together are the group of formula (II), R₄        is F;

R₁, R₂, R₃ and R₄ are linked to the carbon atoms in 17, 16, 6 and 9 ofthe steroidal structure in position α or β;

R is:

wherein

Y is selected from:

1) —R₅—CH(ONO₂)R₆

2) —R—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

4) —[(CH₂)_(o)—(X)]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein

R₅ is a straight or branched C₁-C₁₀ alkylene; preferably R₅ is astraight C₁-C₆ alkylene;

R₆ is H or a straight or branched C₁-C₆ alkyl, preferably R₆ is H or—CH₃;

R₇ and R₈ at each occurrence are independently H or a straight orbranched C₁-C₆ alkyl; preferably R₇ and R₈ at each occurrence areindependently H or CH₃;

R₉ is H or a straight or branched C₁-C₆ alkyl, preferably R₉ is H or—CH₃;

n is an integer from 0 to 6; preferably n is 0 or 1;

o is an integer from 1 to 6; preferably o is an integer from 2 to 4,more preferably o is 2;

p is an integer from 1 to 6; preferably p is an integer from 1 to 4;more preferably p is 1 or 2;

q is an integer from 0 to 6; preferably q is from 0 to 4, morepreferably is 0 or 1;

X is O, S or NR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is O;

with then proviso that the invention does not include the compounds offormula (I) wherein R₁ and R₂ taken together are the group of formula(II)

R₄ is F and R₃ is a hydrogen atom, and R is the compound of formula(III) wherein and Y is —R₅—CH(ONO₂)R₆ and R₆ is H.

In another embodiment of the invention, there is provided a compound offormula (I)

wherein

R₄ is F, R₃ is F, and

R₁ and R₂ taken together are the group of formula (II)

R₁, R₂, R₃ and R₄ are linked to the carbon atoms in 17, 16, 6 and 9 ofthe steroidal structure in position α;

R is:

wherein

Y is selected from:

1) —R₅—CH(ONO₂)R₆

2) —R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

4) —[(CH₂)_(o)—(X)]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein

R₅ is a straight or branched C₁-C₁₀ alkylene; preferably R₅ is astraight C₁-C₆ alkylene;

R₆ is H or a straight or branched C₁-C₆ alkyl, preferably R₆ is H or—CH₃;

R₇ and R₈ at each occurrence are independently H or a straight orbranched C₁-C₆ alkyl; preferably R₇ and R₈ at each occurrence areindependently H or CH₃;

R₉ is H or a straight or branched C₁-C₆ alkyl, preferably R₉ is H or—CH₃;

n is an integer from 0 to 6; preferably n is 0 or 1;

o is an integer from 1 to 6; preferably o is an integer from 2 to 4,more preferably o is 1;

p is an integer from 1 to 6; preferably p is an integer from 1 to 4;more preferably p is 1 or 2;

q is an integer from 0 to 6; preferably q is from 0 to 4, morepreferably is 0 or 1;

X is O, S or NR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is 0.

In another aspect of the invention, there is provided a compound offormula (I)

wherein

R₁ and R₂ taken together are the group of formula (II)

R₄ is F and R₃ is a hydrogen atom;

R₁, R₂ and R₄ are linked to the carbon atoms in 17, 16 and 9 of thesteroidal structure in position α;

R is:

wherein

Y is selected from:

1) —R₅—CH(ONO₂)R₆

2) —R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

4) —[(CH₂)_(o)—(X)]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein

R₅ is a straight or branched C₁-C₁₀ alkylene; preferably R₅ is astraight C₁-C₆ alkylene;

R₆ is a straight or branched C₁-C₆ alkyl, preferably R₆ is —CH₃;

R₇ and R₈ at each occurrence are independently H or a straight orbranched C₁-C₆ alkyl; preferably R₇ and R₈ at each occurrence areindependently H or CH₃;

R₉ is H or a straight or branched C₁-C₆ alkyl, preferably R₉ is H or—CH₃;

n is an integer from 0 to 6; preferably n is 0 or 1;

o is an integer from 1 to 6; preferably o is an integer from 2 to 4,more preferably o is 2;

p is an integer from 1 to 6; preferably p is an integer from 1 to 4;more preferably p is 1 or 2;

q is an integer from 0 to 6; preferably q is from 0 to 4, morepreferably is 0 or 1;

X is O, S or NR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is O.

In another aspect of the invention, there is provided a compound offormula (I)

wherein

R₁ is OH, R₂ is CH₃, R₃ is a hydrogen atom and R₄ is F;

R₁ and R₄ are linked to the carbon atoms 17 and 9 of the steroidalstructure in position α, R₂ is linked to the carbon atom 16 of thesteroidal structure in position β;

R is:

wherein

Y is selected from:

1) —R₅—CH(ONO₂)R₆

2) —R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

4) —[(CH₂)_(o)—(X)]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein

R₅ is a straight or branched C₁-C₁₀ alkylene; preferably R₅ is astraight C₁-C₆ alkylene;

R₆ is H or a straight or branched C₁-C₆ alkyl, preferably R₆ is H or—CH₃;

R₇ and R₈ at each occurrence are independently H or a straight orbranched C₁-C₆ alkyl; preferably R₇ and R₈ at each occurrence areindependently H or CH₃;

R₉ is H or a straight or branched C₁-C₆ alkyl, preferably R₉ is H or—CH₃;

n is an integer from 0 to 6; preferably n is 0 or 1;

o is an integer from 1 to 6; preferably o is an integer from 2 to 4,more preferably o is 2;

p is an integer from 1 to 6; preferably p is an integer from 1 to 4;more preferably p is 1 or 2;

q is an integer from 0 to 6; preferably q is from 0 to 4, morepreferably is 0 or 1;

X is O, S or NR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is O.

In another aspect of the invention, there is provided a compound offormula (I)

wherein

R₁ is OH, R₂ is CH₃, R₃ is a hydrogen atom and R₄ is Cl;

R₁ and R₄ are linked to the carbon atoms 17 and 9 of the steroidalstructure in position α, R₂ is linked to the carbon atom 16 of thesteroidal structure in position β;

R is:

wherein

Y is selected from:

1) —R₅—CH(ONO₂)R₆

2) —R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

4) —[(CH₂)_(o)—(X)]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein

R₅ is a straight or branched C₁-C₁₀ alkylene; preferably R₅ is astraight C₁-C₆ alkylene;

R₆ is H or a straight or branched C₁-C₆ alkyl, preferably R₆ is H or—CH₃;

R₇ and R₈ at each occurrence are independently H or a straight orbranched C₁-C₆ alkyl; preferably R₇ and R₈ at each occurrence areindependently H or CH₃;

R₉ is H or a straight or branched C₁-C₆ alkyl, preferably R₉ is H or—CH₃;

n is an integer from 0 to 6; preferably n is 0 or 1;

o is an integer from 1 to 6; preferably o is an integer from 2 to 4,more preferably o is 2;

p is an integer from 1 to 6; preferably p is an integer from 1 to 4;more preferably p is 1 or 2;

q is an integer from 0 to 6; preferably q is from 0 to 4, morepreferably is 0 or 1;

X is O, S or NR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is O.

Preferred R are:

In another aspect of the invention, there is provided a compoundselected from the following group:

In another aspect of the invention, there is provided a compound offormula (I) for treating inflammatory diseases.

In another aspect of the invention, there is provided a compound offormula (I) for treating ocular diseases, in particular diabetic macularedema, diabetic retinopathy, macular degeneration, age-related maculardegeneration and other diseases of retina and macula lutea. A preferredcompound is the compound of formula (1) above reported.

In another aspect of the invention, there is provided a compound offormula (I) including the compounds of formula (I) wherein R₁ and R₂taken together are the group of formula (II)

R₄ is F and R₃ is a hydrogen atom, and R is the compound of formula(III) wherein and Y is —R₅—CH(ONO₂)R₆ and R₆ is H, for the use in theprevention or in the treatment of diabetic macular edema, diabeticretinopathy, macular degeneration, age-related macular degeneration andother diseases of retina and macula lutea, in particular diabeticmacular edema. A preferred compound is the compound of formula (18)

In yet another aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of formula (I) and/or a salt or stereoisomerthereof and at lest an ophthalmically acceptable excipient in a suitableform for intravitreal or periorbital administration.

The term “excipient” is used herein to describe any ingredient otherthan the compound(s) of the invention. The choice of the excipient willto a large extent depend on factors such as the particular mode ofadministration, the effect of the excipient on the stability, and thenature of the dosage form.

In still another aspect of the invention, there is provided apharmaceutical composition wherein the compound of the invention isadministered as a suspension or emulsion in an ophthalmically acceptablevehicle.

The compounds of the invention intended for pharmaceutical use may beadministered as crystalline or amorphous products. The compounds of theinvention intended for pharmaceutical use may be administered alone orin combination with one or more other compounds of the invention.

The utility of the compounds of the invention as medical agents for thetreatment or prevention of diabetic macula edema, diabetic retinopathy,macular degeneration, age-related macular degeneration and otherdiseases of retina and macula lutea is demonstrated by the activity ofthe compounds in conventional assays.

Synthesis Procedure

1) The compound of general formula (I) wherein R₁, R₂, R₃ and R₄ are asabove defined, the radical R is as defined in formulae III and IVwherein Y is as above defined, can be obtained:

1.1) by reacting a compound of formula (V),

wherein R₁, R₂, R₃, R₄ are as above defined, W is H or C(O)—Cl with acompound of formula (VII) or (VIII)

wherein Y is as above defined and

W₁ is H when W is —C(O)—Cl or

W₁ is —C(O)—Cl or —CO—O—R_(a) when W is H, wherein R_(a) ispentafluorophenyl or 4-nitrophenyl, P₁ is a diol protecting group suchas acetal, such as p-methoxybenzylidene, butylidene, and those describedin T. W. Greene “Protective groups in organic synthesis”, HarvardUniversity Press, 1980, 2^(nd) edition.

1.1.a) the reaction of a compound of formula (V) wherein W is H with acompound of formula (VII) or (VIII) wherein W₁ is —C(O)—Cl, or thereaction of a compound of formula (V) wherein W is —C(O)—Cl with acompound of formula (VII) or (VIII) wherein W₁ is H, may be carried outin presence of an organic base such as N,N-dimethylamino pyridine(DMAP), triethylamine, pyridine. The reaction is carried out in an inertorganic solvent such as N,N′-dimethylformamide, tetrahydrofuran,benzene, toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at atemperature from −20° C. and 40° C. The reaction is completed within atime range from 30 minutes to 36 hours

1.1.b) the reaction of a compound of formula (V) wherein W is H with acompound of formula (VII) or (VIII) wherein W₁ is —C(O)—O—R_(a), whereinR_(a) is as above defined, may be carried out in presence of a catalyst,such as DMAP or in the presence of DMAP and a Lewis acid such asSc(OTf)₃ or Bi(OTf)₃. The reaction is carried out in an inert organicsolvent such as N,N′-dimethylformamide, tetrahydrofuran, benzene,toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at atemperature from −20° C. and 40° C. The reaction is completed within atime range from 30 minutes to 36 hours;

1.2) optionally deprotecting the compounds obtained in step 1.1.a) or1.1.b) according to methods described in T. W. Greene “Protective groupsin organic synthesis”, Harvard University Press, 1980, 2^(nd) edition.Hydrochloric acid in tetrahydrofurane is the preferred method forremoving acetal protecting group.

Preparation of the Compounds of Formula (V)

2) The compounds of formula (V) wherein:

W is H and R₁ is OH, R₂ is CH₃, R₃ is a hydrogen atom, R₄ is F or Cl;

or W is H, R₃ is a hydrogen atom or F, R₄ is F and R₁ and R₂ takentogether are the group of formula (II)

are commercially available.

2.1) The compounds of formula (V) wherein W is —C(O)—Cl or —CO—O—R_(a)and R₁, R₂, R₃ and R₄ are as above defined can be obtained from thecorresponding commercially available compounds of formula (V) wherein Wis H using methods known in the literature.

Preparation of the Compounds of Formula (VII) or (VIII)

3) The compounds of formula (VII) or (VIII) wherein W₁ is H, P₁ is asabove defined and

Y is:

—R₅—CH(ONO₂)R₆

—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉

wherein R₅, R₆, R₉, o, p and q are as above defined can be prepared asfollows:

3.1.a) by reacting a compound of formula (IX) or (X),

wherein P is a hydroxyl protecting group such as silyl ethers, such astrimethylsilyl, tert-butyl-dimethylsilyl or trityl and those describedin T. W. Greene “Protective groups in organic synthesis”, HarvardUniversity Press, 1980 2^(nd) edition, P₁ is as above reported,

with a compound of formula (XI) or (XII)

HO—R₅—CH(Q)R₆ or   (XI)

HO—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(Q)R₉   (XII)

wherein R₅, R₆, R₉, o, p and q are as above defined and Q is ONO₂ or Q₁,wherein Q₁ is a chlorine atom, a bromine atom, a iodine atom, a mesylgroup or a tosyl group,

in the presence of a condensing agent such as dicyclohexylcarbodiimide(DCC), N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(EDAC), N,N′-carbonyldiimidazole (CDI), optionally in the presence of abase, for example DMAP.

The reaction is carried out in an inert organic solvent dry such asN,N′-dimethylformamide, tetrahydrofuran, benzene, toluene, dioxane, apolyhalogenated aliphatic hydrocarbon at a temperature from −20° C. and50° C. The reaction is completed within a time range from 30 minutes to36 hours;

3.1.b) by reacting a compound of formula (IX) or (X) as above reportedwith a compound of formula (XIII) or (XIV)

W₃—R₅—CH(Q)R₆ or   (XIII)

W₃—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(Q)R₉   (XIV)

wherein R₅, R₆ R₉, o, p, q and Q are as above defined and W₃ is Cl, Br,I, in the presence of a organic base such as1,8-diazabiciclo[5.4.0]undec-7-ene (DBU), N,N-diisopropylethyl amine,diisopropylamine or an inorganic base such as alkaline-earth metalcarbonate or hydroxide, potassium carbonate, cesium carbonate, in aninert organic solvent such as N,N′-dimethylformamide, tetrahydrofuran,acetone, methyl ethyl ketone, acetonitrile, a polyhalogenated aliphatichydrocarbon at a temperature from −20° C. and 40° C., preferably from 5°C. to 25° C. The reaction is completed within a time range from 1 tohours. When W₃ is chosen among chlorine or bromine the reaction iscarried out in presence an iodine salts such as KI.

3.1.c) by reacting a compound of formula (IXa) or (Xa)

wherein P and P₁ are as above defined, and R_(b) is pentafluorophenyl,4-nitrophenyl, or —(N-succimidyl), with a compound of formula (XI) or(XII)

HO—R₅—CH(Q)R₆ or   (XI)

HO—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(Q)R₉   (XII)

wherein R₅, R₆, R₉, o, p, q and Q are as above defined, in the presenceof a catalyst, such as DMAP or in the presence of DMAP and a Lewis acidsuch as Sc(OTf)₃ or Bi(OTf)₃. The reaction is carried out in an inertorganic solvent such as N,N′-dimethylformamide, tetrahydrofuran,benzene, toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at atemperature from −20° C. and 40° C. The reaction is completed within atime range from 30 minutes to 36 hours;

3.1.d) by reacting a compound of formula (IXb) or (Xb)

with a compound of formula (XI) or (XII)

HO—R₅—CH(Q)R₆ or   (XI)

HO—[(CH₂)_(c)—X]_(p)—(CH₂)_(q)—CH(Q)R₉   (XII)

wherein R₅, R₆, R₉, o, p, q and Q are as above defined, in the presenceof an organic base such as N,N-dimethylamino pyridine (DMAP),triethylamine, pyridine. The reaction is carried out in an inert organicsolvent such as N,N′-dimethylformamide, tetrahydrofuran, benzene,toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at atemperature from −20° C. and 40° C. The reaction is completed within atime range from 30 minutes to 36 hours.

3.2) by reacting the compound obtained in the steps 3.1.a)-3.1.d)wherein Q is Q₁, with a nitrate source such as silver nitrate, lithiumnitrate, sodium nitrate, potassium nitrate, magnesium nitrate, calciumnitrate, iron nitrate, zinc nitrate or tetraalkylammonium nitrate(wherein alkyl is C₁-C₁₀ alkyl) in a suitable organic solvent such asacetonitrile, tetrahydrofurane, methyl ethyl ketone, ethyl acetate, DMF,the reaction is carried out, in the dark, at a temperature from roomtemperature to the boiling temperature of the solvent. Alternatively thereaction with AgNO₃ can be performed under microwave irradiation insolvents such acetonitrile or THF at temperatures in the range betweenabout 100-180° C. for time range about 1-60 min. Preferred nitratesource is silver nitrate and

3.3) optionally removing the hydroxyl protective group P according tothe methods described in T. W. Greene “Protective groups in organicsynthesis”, Harvard University Press, 1980, 2^(nd) edition. Fluoride ionis the preferred method for removing the silyl ether group.

4) The compounds of formula (VII) or (VIII) wherein W₁ is H, P₁ is asabove defined and

Y is

—R₅—CH(ONO₂)—(CR₇R₈)_(p)—CH(ONO₂)R₉

—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein R₅, R₉, R₇, R₈, o, p and q are as above defined and n is 0 canbe prepared as follows:

4.1.a) by reacting a compound of formula (IX) or (X) as above reported,with a compound of formula (XV) or (XVI)

HO—R₅—CH═CH—R₉   (XV)

HO—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH═CH—R₉   (XVI)

wherein R₅, o, p, q, X and R₉ are as above defined,

according to the method described in 3.1.a);

4.1.b) by reacting a compound of formula (IX) or (X) as above reported,

with a compound of formula (XVII) or (XVIII)

W₃—R₅—CH═CH—R₉   (XVII)

W₃—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH═CH—R₉   (XVIII)

wherein R₅, o, p, q, X and R₉ are as above defined and W₃ is Cl, Br, I,according to the method described in 3.1.b)

4.1.c) by reacting a compound of formula (IXa) or (Xa) as abovereported,

with a compound of formula (XV) or (XVI)

HO—R₅—CH═CH—R₉   (XV)

HO—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH═CH—R₉   (XVI)

wherein R₅, o, p, q, X and R₉ are as above defined, according to themethod described in 3.1.c)

4.1.d) by reacting a compound of formula (IXb) or (Xb) as above reportedwith a compound of formula (XV) or (XVI)

HO—R₅—CH═CH—R₉   (XV)

HO—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH═CH—R₉   (XVI)

wherein R₅, o, p, q, X and R₉ are as above defined, according to themethod described in 3.1.d)

4.2.a) by reacting a compound of formula (VIIA) or (VIIIA)

wherein P and P₁ are as above defined and Y′ is:

—R₅—CH═CH—R₉

—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH═CH—R₉

wherein R₅, o, p, q and R₉ are as above defined,

with a nitrate source such as silver nitrate, in presence of iodine in asuitable organic solvent such as acetonitrile, tetrahydrofurane, methylethyl ketone, ethyl acetate, DMF, the reaction is carried out, in thedark, at a temperature from −20° C. to the boiling temperature of thesolvent. Alternatively the reaction can be performed under microwaveirradiation in solvents such acetonitrile or THF at temperatures in therange between about 100-180° C. for time range about 1-60 min.Alternatively

4.2.b) by dihydroxylation of the double bond of the compound of formula(VIIA) or (VIIIA) above defined to obtain a compound (VIIB) or (VIIIB)

wherein P and P₁ are as above defined and Y″ is:

—R₅—CH(OH)—CH(OH)—R₉

—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(OH)—CH(OH)—R₉

wherein R₅, o, p, q and R₉ are as above defined,

with a reagent for Sharpless asymmetric dihydroxylation such as ADmixalpha or ADmix beta in a mixture water/tert-butanol, at a temperaturefrom −20° C. and 30° C., preferably from −5° C. to 5° C. The reaction iscompleted within a time range from 1 to 24 hours.

4.3) by reacting the compound obtained in the steps 4.2.b) with nitricacid and acetic anhydride in a suitable organic solvent such asmethylene chloride, in a temperature range from −50° C. to 0° C.according to methods well known in the literature.

4.4) optionally deprotecting the compounds obtained in step 4.2.a) or4.3) as described in T. W. Greene “Protective groups in organicsynthesis”, Harvard University Press, 1980, 2^(nd) edition. Fluoride ionis the preferred method for removing silyl ether protecting group.

5) The compounds of formula (VII) or (VIII) wherein W₁ is H, P₁ is asabove defined and Y is

—R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

—[(CH₂)_(n)—X]_(p)—(CH₂)_(q)—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉

wherein R₅, R₉, R₇, R₈, o, p and q are as above defined and n is aninteger from 1 to 6 can be prepared as follows:

5.1) by reacting a compound of formula (IX) or (X) as above reportedwith a compound of formula (XIX) or (XX)

W₃—R₅—CH(Q₂)—(CR₇R₈)_(n)—CH (Q₂)R₉ or   (XIX)

W₃—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(Q₂)—(CR₇R₈)_(n)—CH(Q₂)R₉   (XX)

wherein R₅, R₉, R₇, R₈, n, o, p and q are as above defined, Q₂ is ONO₂or OH and W₃ is Cl, Br, I, according to the method described in 3.1.b);

5.2) by reacting the compound obtained in the steps 5.1) wherein Q isOH, with a nitrate source according to the method described in 4.3);

5.3) optionally removing the hydroxyl protective group P according tothe method described in 3.3);

Preparation of compounds (IX), (IXa), (IXb), (X), (Xa) and (Xb)

5) The compounds of formula (IX), (IXa), (IXb), (X), (Xa) and (Xb)wherein P, P₁ and R_(b) are as above described, can be prepared startingfrom vanillic acid or gallic acid, which are commercially available,according to method known in the literature.

Preparation of compounds (XI)-(XX)

6.1) The compounds of formula (XI)-(XIV) wherein R₅, R₆, R₉, o, p, q andW₃ are as above defined and Q is Q₁ wherein Q₁ is as above defined, arecommercially available or can be obtained according methods known in theliterature.

6.2) The compounds of formula (XI)-(XIV) wherein R₅, R₆, R₉, o, p, q andW₃ are as above defined and Q is ONO₂ can be obtained from thecorresponding compounds wherein Q is Q₁ by reaction with a nitratesource as above described.

6.3) The compounds of formula (XV)-(XX) wherein W₃, R₅, R₉, R₇, R₆, n,o, p and q are as above defined, are commercially available or can beobtained according to methods known in the literature.

EXAMPLE 1 Compound (1)

Synthesis of 4-(nitrooxy)butyl4-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-16,17-(1-methylethylidenebis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

A) 4-(Nitrooxy)butyl 4-hydroxy-3-methoxybenzoate

To a solution of vanillic acid (5.0 g, 29.73 mmol) inN,N-dimethylformamide (50 ml), cesium carbonate (9.68 g, 29.73 mmol) wasadded. The reaction was cooled at 0° C. and a 20% solution of1-bromo-4-(nitrooxy)butane in dichloromethane (29.45 g) was added. Thereaction was stirred at room temperature for 69 hours. The mixture waspoured into a 5% aqueous NaH₂PO₄ solution and extracted with diethylether (3×70 ml) The organic layers were washed with water (70 ml), driedover sodium sulfate and concentrated under reduced pressure. The residuewas purified by flash chromatography (Biotage System, Cartridge columnFLASH 65+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 95/5 (500ml), to n-hexane/ethyl acetate 1/1 during 4000 ml, n-hexane/ethylacetate 1/1 (500 ml)). The product (5.88 g) was obtained.

B) 4-(Nitrooxy)butyl-3-methoxy-4-((4-nitrophenoxy)carbonyloxy)benzoate

To a solution of compound A (2.94 g, 10.30 mmol) in dichloromethane (50ml), cooled at 0° C., pyridine (1.01 ml, 10.30 mmol) and p-nitrophenylchloroformate (2.07 ml, 10.30 mmol) were added. The reaction was stirredat room temperature for 16 hours. The mixture was washed with 1M aqueousHCl (2×50 ml), the organic layer was dried over sodium sulfate andconcentrated under reduced pressure. The residue was purified by flashchromatography (Biotage System, Cartridge column FLASH 65+M™ KP-Sil,eluent: gradient n-hexane/ethyl acetate 93/7 (500 ml), to n-hexane/ethylacetate 1/1 during 4000 ml, n-hexane/ethyl acetate 1/1 (500 ml)). Theproduct (3.50 g) was obtained.

C) 4-(nitrooxy)butyl4-((2-((6S,9R,10S,11S,135,16R,17S)-6,9-difluoro-11-hydroxy-16,17-16,17-(1-methylethylidenebis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

To a solution of compound B (1.00 g, 2.28 mmol) in chloroform (30 ml),scandium triflate (0.10 g, 0.22 mmol) and DMAP (0.54 g, 4.56 mmol) wereadded. The reaction was cooled at 0° C. and fluocinolone acetonide (0.99g, 2.28 mmol) was added. The reaction was stirred at room temperaturefor 28 hours. The mixture was diluted with dichloromethane (30 ml),washed with 5% NaH₂PO₄ and then with saturated aqueous sodium carbonate.The organic layer was dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by flash chromatography,(Biotage System, column FLASH 40+M™ KP-Sil, eluent: gradientn-hexane/ethyl acetate 9/1 (200 ml), to n-hexane/ethyl acetate 3/7during 2000 ml, n-hexane/ethyl acetate 3/7 (600 ml)). The product (0.29g) was obtained.

The product was crystallized by n-hexane/ethyl acetate

M.p.=199-200° C.

¹H-NMR: (DMSO), δ: 7.65 (2H, d); 7.38 (1H, d); 7.27 (1H, d); 5.60 (1H,dm); 5.50 (1H, s); 5.35 (2H, m); 4.60 (2H, t); 4.35 (2H, t); 4.20 (1H,m); 3.89 (3H, s); 2.75-2.50 (2H, m); 2.25 (1H, m); 2.00 (2H, m);1.90-1.30 (13H, m); 1.15 (3H, s); 0.83 (3H, s).

EXAMPLE 2 Compound (3) Synthesis of 5,6-bis(nitrooxy)hexyl4-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

D) Hex-5-enyl 4-hydroxy-3-methoxybenzoate

To a solution of vanillic acid (0.6 g, 3.56 mmol) inN,N-dimethylformamide (7 ml), diisopropylethylamine (0.93 ml, 5.35 mmol)and 6-bromohex-1-ene (0.71 ml, 5.35 mmol) were added. The reaction wasstirred at 50° for 8 hours. The solvent was evaporated under vacuum. Theresidue was purified by flash chromatography (Biotage System, columnFLASH 40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 95/5 (200ml), to n-hexane/ethyl acetate 7/3 during 2000 ml, n-hexane/ethylacetate 3/7 (400 ml)). The product (0.59 g) was obtained.

E) Hex-5-enyl 4-(tert-butyldimethylsilyloxy)-3-methoxybenzoate

To a solution of compound D (1.16 g, 4.64 mmol) in N,N-dimethylformamide(30 ml), imidazole (1.18 g, 17.40 mmol) andtert-butyldimethylchlorosilane (1.31 g, 8.7 mmol) were added. Thereaction was stirred at room temperature for 12 hours. The mixture waspoured in water (50 ml) solution and extracted with diethyl ether (3×50ml) The organic layers were dried over sodium sulfate and concentratedunder reduced pressure. The residue was purified by flash chromatography(Biotage System, column FLASH 40+M™ KP-Sil, eluent: n-hexane/ethylacetate 95/5. The product (1.56 g) was obtained.

F)5,6-Bis(nitrooxy)hexyl-4-(tert-butyldimethylsilyloxy)-3-methoxybenzoate

To a solution of compound E (1.4 g, 3.97 mmol) in acetonitrile (30 ml),silver nitrate (0.8 g, 4.77 mmol) was added. The reaction was cooled at−15° C. and iodine (1.21 g, 4.77 mmol) was added. The reaction wasstirred at −15° C. for 20 minutes. The temperature was risen to 25° C.and iodine (2.7 g, 15.9 mmol) was added. The reaction was heated to 100°C. for 60 minutes under microwave irradiation. The resulting mixture wascooled, filtered and the solvent was removed under reduced pressure. Theresidue was purified by flash chromatography (Biotage System, columnFLASH 40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 9/1 (400ml), to n-hexane/ethyl acetate 7/3 during 2000 ml, n-hexane/ethylacetate 7/3 (400 ml)). The product (1.19 g) was obtained.

G) 5,6-Bis(nitrooxy)hexyl 4-hydroxy-3-methoxybenzoate

To a solution of compound F (1.19 g, 2.43 mmol) in tetrahydrofurane (40ml) cooled at −0° C., a solution of tetrabutylamonium floride 1M intetrahydrofurane (2.43 ml, 2.43 mmol) was added. The reaction wasstirred at 0° C. for 20 minutes. The mixture was poured into a 5%aqueous NaH₂PO₄ solution and extracted with ethyl acetate (3×50 ml) Theorganic layers were washed with water (50 ml), dried over sodium sulfateand concentrated under reduced pressure. The residue was purified byflash chromatography (Biotage System, column FLASH 40+M™ KP-Sil, eluent:gradient n-hexane/ethyl acetate 9/1 (200 ml), to n-hexane/ethyl acetate1/1 during 1000 ml, n-hexane/ethyl acetate 1/1 (200 ml), ton-hexane/ethyl acetate 4/6 during 200 ml, n-hexane/ethyl acetate 4/6(400 ml)). The product (0.9 g) was obtained.

H)2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethylcarbonochloridate

To a solution of fluocinolone acetonide (1.2 g, 2.65 mmol) intetrahydrofurane (24 ml), cooled at 0° C. and under N₂, a 20% toluenesolution of phosgene (5.58 ml, 10.6 mmol) was added. The reaction wasstirred at 0° C. for 30 minutes and at room temperature for 12 hours.The excess of phosgene was removed by heating at 40° C. for 45 minutes.The solvent was evaporated under vacuum. The crude product was used inthe next step without any purification.

I) 5,6-Bis(nitrooxy)hexyl4-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

To a solution of compound H (0.56 g, 1.09 mmol) in dichloromethane (24ml), diisopropylethylamine (0.21 ml, 1.2 mmol) was added. The reactionwas cooled at 0° C. and a solution of compound G (0.45 g, 1.2 mmol) indichloromethane (3 ml) was added. The reaction was stirred at roomtemperature for 12 hours. The solvent was evaporated under vacuum. Theresidue was purified by flash chromatography (Biotage System, columnFLASH 40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 8/2 (200ml), to n-hexane/ethyl acetate 2/8 during 2400 ml, n-hexane/ethylacetate 2/8 (400 ml)). The product (0.67 g) was obtained.

¹H-NMR: (CDCl₃), δ: 7.70 (2H, d); 7.30 (1H, d); 7.07 (1H, d); 6.45 (1H,s); 6.38 (1H, dd); 5.52-5.28 (2H, m); 5.16-4.91 (2H, dd); 5.04 (1H, d);4.74 (1H, dd); 4.50 (1H, m); 4.43-4.35 (3H, m); 3.95 (3H, s); 2.60-2.10(4H, m); 1.90-1.47 (16H, m); 1.25 (3H, s); 0.95 (3H, s).

EXAMPLE 3 Compound (5) Synthesis of2-(2-(2-(nitrooxy)ethoxy)ethoxy)ethyl4-((2-((6S,9R,105,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

J) 4-(tert-butyldimethylsilyloxy)-3-methoxybenzoic acid

To a solution of vanillic acid (2.0 g, 11.89 mmol) inN,N-dimethylformamide (50 ml), imidazole (4.04 g, 59.45 mmol) andtert-butyldimethylchlorosilane (4.48 g, 29.72 mmol) were added. Thereaction was stirred at room temperature for 24 hours. he mixture waspoured in water (70 ml) solution and extracted with diethyl ether (3×70ml) The organic layers were dried over sodium sulfate and concentratedunder reduced pressure. The residue was purified by flash chromatography(Biotage System, column FLASH 40+M™ KP-C18 HS, eluent: gradientacetonitrile/water 65/35 (600 ml), to acetonitrile/water 80/20 during1200 ml). The product (0.70 g) was obtained.

K) 2-(2-(2-chloroethoxy)ethoxy)ethyl-4-(tert-butyldimethylsilyloxy)-3-methoxybenzoate

To a solution of compound J (1.25 g, 4.42 mmol) in dichloromethane (60ml), 2-(chloroethoxy)-ethoxy ethanol (0.83 g, 5.75 mmol) and DMAP (cat.amount) were added. The reaction was cooled at 0° C. and EDAC (1.10 g,5.75 mmol) was added. The reaction was stirred at room temperature for12 hours. The solvent was evaporated under vacuum. The residue waspurified by flash chromatography (Biotage System, Cartridge column FLASH40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 9/1 (40 ml), ton-hexane/ethyl acetate 6/4 during 2000 ml, n-hexane/ethyl acetate 6/4(400 ml)). The product (1.25 g) was obtained.

L) 2-(2-(2-nitrooxyethoxy)ethoxy)ethyl 4-(tert-butyldimethylsilyloxy)-3-methoxybenzoate

To a solution of compound K (1.53 g, 3.54 mmol) in acetonitrile (45 ml),sodium iodide (3.18 g, 21.24 mmol) was added. The reaction was heated to120° C. for 20 minutes under microwave irradiation. The resultingmixture was cooled, filtered and the solvent was removed under reducedpressure. To a solution of residue in acetonitrile (45 ml), silvernitrate (2.04 g, 14.16 mmol) was added. The reaction was heated to 120°C. for 5 minutes under microwave irradiation. The resulting mixture wascooled, filtered and the solvent was removed under reduced pressure. Theresidue was purified by flash chromatography (Biotage System, columnFLASH 40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 9/1 (600ml), to n-hexane/ethyl acetate 6/4 during 2000 ml, n-hexane/ethylacetate 6/4 (400 ml)). The product (1.37 g) was obtained.

M) 2-(2-(2-nitrooxyethoxy)ethoxy)ethyl 4-hydroxy-3-methoxy benzoate

To a solution of compound L (1.10 g, 2.4 mmol) in tetrahydrofurane (40ml) cooled at −0° C., a solution of tetrabutylamonium floride 1M intetrahydrofurane (2.4 ml, 2.4 mmol) was added. The reaction was stirredat 0° C. for 20 minutes. The mixture was poured into a 5% aqueousNaH₂PO₄ solution (100 ml) and extracted with ethyl acetate (3×50 ml) Theorganic layers were washed with water (100 ml), dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified by flash chromatography (Biotage System, column FLASH 40+M™KP-Sil, eluent: gradient n-hexane/ethyl acetate 9/1 (600 ml), ton-hexane/ethyl acetate 1/1 during 2000 ml, n-hexane/ethyl acetate 1/1(400 ml)). The product (0.76 g) was obtained.

N) 2-(2-(2-(nitrooxy)ethoxy)ethoxy)ethyl4-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

To a solution of compound H (0.508 g, 0.98 mmol) in dichloromethane (15ml), diisopropylethylamine (0.18 ml, 1.06 mmol) was added. The reactionwas cooled at 0° C. and a solution of compound M (0.37 g, 1.08 mmol) indichloromethane (3 ml) was added. The reaction was stirred at roomtemperature for 12 hours. The solvent was evaporated under vacuum. Theresidue was purified by flash chromatography (Biotage System, columnFLASH 40+M™ KP-Sil, eluent: gradient n-hexane/ethyl acetate 8/2 (200ml), to ethyl acetate 100% during 2400 ml, ethyl acetate 100% (400 ml)).The product (0.70 g) was obtained.

¹H-NMR: (CDCl₃), δ: 7.70 (2H, d); 7.30 (1H, d); 7.07 (1H, d); 6.45 (1H,s); 6.38 (1H, dd); 5.52-5.32 (1H, m); 5.15-4.91 (2H, dd); 5.04 (1H, d);4.57-4.49(4H, m) : 4.41 (1H, m); 3.95 (3H, s); 3.84 (2H, dd); 3.78 (2H,dd); 3.68(4H, m); 2.60-2.10 (4H, m); 1.90-1.47 (10H, m); 1.25 (3H,s);0.95 (3H, s).

EXAMPLE 4 Compound (6) Synthesis of 4-(nitrooxy)butyl3-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-4,5-dihydroxybenzoate

O) Methyl7-hydroxy-2-(4-methoxyphenyl)benzo[d][1,3]dioxole-5-carboxylate

To a suspension of methyl gallate (10 g, 54.3 mmol) in toluene (25 ml),p-toluensulfonic acid (29 mg) and p-anisaldehyde dimethylacetal (11.56ml, 67.88 mmol) were added. The reaction was refluxed for 1.5 hours withcontinuous removal of water. The mixture was diluted withdichloromethane (70 ml) and washed with a saturated aqueous NaHCO₃solution (100 ml) and extracted with ethyl acetate (3×50 ml) The organiclayer was washed with water (100 ml), dried over sodium ulphate andconcentrated under reduced pressure. The residue was crystallized byn-hexane. The product (8.6 g) was obtained.

P) 7-Hydroxy-2-(4-methoxyphenyl)benzo[d][1,3]dioxole-5-carboxylic acid

To a suspension of compound O (8.6 g, 28.41 mmol) in water/ethanol 5/95(260 ml), sodium hydroxide (2.5 ml, 62.5 mmol) was added. The reactionwas refluxed for 15 hours The solvent was evaporated under vacuum. Theresidue was dissolved in water (150 ml) and extracted with ethyl acetate(100 ml). The aqueous layer was acidified to pH 4 with 1N aqueous HCland extracted with ethyl acetate (6×50 ml) The organic layers were driedover sodium ulphate and concentrated under reduced pressure. The crudeproduct (5.78 g) was used in the next step without any purification.

Q)4-(nitrooxy)butyl-7-hydroxy-2-(4-methoxyphenyl)benzo[d][1,3]dioxole-5-carboxylate

To a solution of compound P (5.78 g, 20.05 mmol) inN,N-dimethylformamide (50 ml), cesium carbonate (6.52 g, 20.05 mmol) wasadded. The reaction was cooled at 0° C. and a 20% solution of1-bromo-4-(nitrooxy)butane in dichloromethane (19.85 g) was added. Thereaction was stirred at room temperature for 40 hours. The mixture waspoured into a 5% aqueous NaH₂PO₄ solution and extracted with diethylether (2×70 ml) The organic layers were washed with water (50 ml), driedover sodium ulphate and concentrated under reduced pressure. The residuewas purified by flash chromatography (Biotage System, SNAP Cartridgesilica 100 g, eluent: gradient n-hexane/ethyl acetate 9/1 (200 ml), ton-hexane/ethyl acetate 1/1 during 1200 ml, n-hexane/ethyl acetate 1/1(400 ml)). The product (4.36 g) was obtained.

R) 4-(Nitrooxy)butyl7-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-2-(4-methoxyphenyl)benzo[d][1,3]dioxole-5-carboxylate

To a solution of compound Q (0.519 g, 1.28 mmol) in dichloromethane (13ml), diisopropylethylamine (0.179 ml, 1.28 mmol) was added. The reactionwas cooled at 0° C. and a solution of compound H (0.6 g, 1.16 mmol) indichloromethane (3 ml) was added. The reaction was stirred at roomtemperature for 16 hours. The solvent was evaporated under vacuum. Theresidue was purified by flash chromatography (Biotage System, SNAPCartridge silica 100 g, eluent: gradient n-hexane/ethyl acetate 9/1 (200ml), to n-hexane/ethyl acetate 3/7 during 1200 ml, n-hexane/ethylacetate 3/7 (400 ml)). The product (0.979 g) was obtained.

4-(Nitrooxy)butyl3-((2-((6S,9R,10S,11S,135,16R,17S)-6,9-difluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-4,5-dihydroxybenzoate

To a solution of compound R (0.97 g, 1.09 mmol) in tetrahydrofurane(22.4 ml), 1N aqueous HCl (22.4 ml) was added. The reaction was stirredat room temperature for 17 hours. The solvent was evaporated undervacuum. The residue was extracted with ethyl acetate (2×30 ml) Theorganic layers were dried over sodium sulphate and concentrated underreduced pressure. The residue was purified by flash chromatography(Biotage System, purified by flash chromatography (Biotage System, SNAPCartridge silica 100 g, eluent: gradient acetone/dichloromethane 5/95(200 ml), to acetone/dichloromethane 2/8 during 900 ml, toacetone/dichloromethane 3/7 during 600 ml). The product (0.344 g) wasobtained.

¹H-NMR: (CDCl₃), δ: 7.51 (2H, dd); 7.14 (1H, d); 6.47 (1H, s); 6.40 (1H,dd); 5.52-5.32 (1H, m); 5.24-4.93 (2H, dd); 5.03 (1H, d); 4.53 (2H, t):4.43-4.33 (3H, m); 2.54-2.17 (4H, m); 2.00-1.65 (8H, m); 1.53 (3H, s);1.47 (3H,s); 1.25 (3H,s); 0.94 (3H, s).

EXAMPLE 5 Compound (10) Synthesis of 4-(nitrooxy)butyl3-((2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-4,5-dihydroxybenzoate

S)2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethylcarbonochloridate

To a solution of triamcinolone acetonide (3 g, 6.9 mmol) intetrahydrofurane (33 ml), cooled at 0° C. and under N₂, a 20% toluenesolution of phosgene (21.8 ml, 41.4 mmol) was added. The reaction wasstirred at 0° C. for 1 hour and at room temperature for 17 hours. Theexcess of phosgene was removed by heating at 40° C. for 30 minutes. Thesolvent was evaporated under vacuum. The crude product was used in thenext step without any purification.

T) 4-(Nitrooxy)butyl7-((2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-2-(4-methoxyphenyl)benzo[d][1,3]dioxole-5-carboxylate

To a solution of compound Q (0.583 g, 1.32 mmol) in dichloromethane (14ml), diisopropylethylamine (0.231 ml, 1.32 mmol) was added. The reactionwas cooled at 0° C. and a solution of compound S (0.6 g, 1.2 mmol) indichloromethane (3 ml) was added. The reaction was stirred at roomtemperature for 16 hours. The solvent was evaporated under vacuum. Theresidue was purified by flash chromatography (Biotage System, SNAPCartridge silica 100 g, eluent: gradient n-hexane/ethyl acetate 9/1 (200ml), to n-hexane/ethyl acetate 3/7 during 1200 ml, n-hexane/ethylacetate 3/7 (400 ml)). The product (0.819 g) was obtained.

U) 4-(Nitrooxy)butyl3-((2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-4,5-dihydroxybenzoate

To a solution of compound T (0.81 g, 0.93 mmol) in tetrahydrofurane(19.5 ml), 1N aqueous HCl (19.5 ml) was added. The reaction was stirredat room temperature for 18 hours. The solvent was evaporated undervacuum. The residue was extracted with ethyl acetate (2×30 ml) Theorganic layers were dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by flash chromatography(Biotage System, purified by flash chromatography (Biotage System, SNAPCartridge silica 100 g, eluent: gradient acetone/dichloromethane 5/95(200 ml), to acetone/dichloromethane 3/7 during 900 ml,acetone/dichloromethane 3/7 (200 ml). The product (0.245 g) wasobtained.

¹H-NMR: (CDCl₃), δ: 7.51 (2H, dd); 7.23 (1H, d); 6.38 (1H, s); 6.18 (1H,dd); 5.21-4.90 (2H, dd); 5.03 (1H, d); 4.53 (2H, t): 4.41-4.32 (3H, m);2.69-2.35 (4H, m); 2.00-1.65 (10H, m); 1.53 (3H, s); 1.45 (3H,s); 1.24(3H,s); 0.94 (3H, s).

EXAMPLE 6 Compound (17) 2-(2-(2-(nitrooxy)ethoxy)ethoxy)ethyl4-((2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-((1-methylethylidene)bis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

The compound was synthesized using the procedure described in example 3starting from compound S and compound M.

¹H-NMR: (CDCl₃), δ: 7.71 (2H, d); 7.26 (1H, d); 7.15 (1H, d); 6.31 (1H,dd); 6.12 (1H, s); 5.12 (1H, d); 4.91 (1H, d); 5.01 (2H, d); 4.56 (2H,m); 4. 49 (2H, t) : 4.40 (1H, m); 3.95 (3H, s); 3.79 (2H, t); 3.76 (2H,m); 3.67 (4H, m); 2.65-2.35 (4H, m); 2.15-2.00 (1H, m); 1.92-1.84 (1H,m); 1.72-1.55 (2H, m); 1.51 (3H, s); 1.45 (3H,s); 1.25 (5H, m); 0.93(3H, s).

EXAMPLE 7 Compound (18) 4-(nitrooxy)butyl4-((2-((9R,10S,11S,13S,16R,17S)-9-fluoro-11-hydroxy-16,17-(1-methylethylidenebis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate

The compound was synthesized using the procedure described in example 1starting from triamcinolone acetonide and compound B.

¹H-NMR: (CDCl₃), δ: 7.65 (2H, m); 7.26 (1H, d); 7.17 (1H, d); 6.40 (1H,dd); 6.10 (1H, s); 5.11-4.84 (2H, dd); 4.99 (1H, d); 4.53 (2H, t); 4.37(2H, t); 3.93 (3H, s); 2.71-2.30 (5H, m); 2.00-1.50 (6H, m); 1.87 (4H,m); 1.50 (3H, s); 1.41 (3H, s); 1.22 (3H, s); 0.92 (3H, s).

EXAMPLE F1 Assay on Vascular Tone

Test Compounds:

Compound (1) described in Ex. 1

Compound (3) described in Ex. 2

Compound (5) described in Ex. 3

Compound (6) described in Ex. 4

Compound (10) described in Ex. 5

Compound (18) described in Ex. 7

Reference Compounds:

Fluocinolone acetonide (FC)

Triamcinolone acetonide (TAAC)

The ability of the compounds of the invention to induce vasorelaxationin comparison to precursor compounds was tested in vitro in isolatedrabbit thoracic aorta preparations (Wanstall J. C. et al., Br. J.Pharmacol., 134:463-472, 2001). Male New Zealand rabbits (1.8-2 Kg) wereused. The animals were anaesthetized with sodium thiopental (50 mg/kg,iv), sacrificed by exsanguinations and then the thorax was opened andthe aorta dissected. The aortas were placed immediately in Krebs-HEPESbuffer (pH 7.4; composition mM: NaCl 130.0, KCl 3.7, NaHCO₃ 14.9, KH₂PO₄1.2, MgSO₄ 7H₂O 1.2, Glucose 11.0, HEPES 10.0, CaCl₂.2H₂O 1.6) and cutinto ring segments (4-5 mm in length). Each ring was placed in a 5 mltissue bath filled with Krebs-HEPES buffer (37° C.) aerated with 95% O₂and 5% CO₂ and was then attached to a force transducer (Grass FT03),connected to a BIOPAC MP150 System for measurement of the isometrictension². The preparations were allowed to equilibrate for 1 h at aresting tension of 2 g with changes of the buffer every 15 minutes andthen stimulated by exposure to 90 mM KCl (3 times) with interveningwashings. After equilibration, the rings were precontracted submaximallywith methoxamine (3 μM) and, when the contraction reach a steady state acumulative concentration-response curve to the test compounds wasobtained. The time intervals between doses were based on the time neededto reach a full a steady state response.

Responses to test compounds were expressed as a percentage of residualcontraction and plotted against concentration of test compound. EC₅₀values (where EC₅₀ is the concentration producing 50% of the maximumrelaxation to the test compound) were interpolated from these plots.

As shown in Table 1, the test compounds were able to induce relaxationin a concentration-dependent manner.

TABLE 1 Assay on vascular tone Test Compound EC₅₀ (μM) FC no effectCompound (1) 2.2 Compound (3) 0.21 ± 0.07 Compound (5) 0.83 ± 0.25Compound (6) 1.44 ± 0.41 TAAC no effect Compound (18) 1.56 Compound (10)2.35 ± 0.98

EXAMPLE F2

Evaluation of the efficacy of the compounds of the invention in an invivo VEGF-induced leakage rat model.

Vascular endothelial growth factor (VEGF) activates common pathways ofvascular leakage associated with various pathological processesincluding diabetic macular edema (DME) Test compounds:

Compound (1) disclosed in Example 1

Fluocinolone acetonide (FC): reference compound of compound (1)

Compound (18) described in Example 7

Triamcinolone acetonide (TAAC): reference compound of compound (18)

Male Sprague Dawley rats (˜250 g; Charles River laboratory) wereanesthetized with isoflurane inhalation and a drop of 0.5% tetracainewas topically applied on the eyes. Pupils were dilated with 1% topicalcyclopentolate hydrochloride to see the needle to guide the intravitrealinjection. Recombinant rat vascular endothelial growth factor (VEGF; 100ng/eye) or VEGF at 100 ng/eye plus test compounds were prepared in 0.5%carboxymethyl cellulose (CMC) in sterile saline and was injected intothe vitreous with a 30-gauge needle (Xu, Q., et al. Invest. Ophthalmol.Vis. Sci., 42:789-794, 2001). The compounds of the invention werecompared to corresponding steroid cores at equimolar doses. For example,to compare to fluocinolone acetonide at both 25 and 50 μg/eye, compound(1) was dosed at 42.4 and 84.7 μg/eye, respectively. Control animalsreceived vehicle. Compound (18) was similarly dosed alongsidetriamcinolone acetonide for comparison.

Retinal vascular leakage was measured as described previously (Xu, Q.,et al. Invest. Ophthalmol. Vis. Sci., 42:789-794, 2001). After about 18hours post injection of test compounds, rats were anesthetized withketamine (80 mg/kg) and xylazine (4 mg/kg) IP. Then 45 mg/kg Evans blue(EB) was injected into the jugular vein. The dye was allowed tocirculate for 2 hours. The chest cavity was opened and rats thenperfused with 1% formalin in 0.5 M citrate buffer (pH 3.5, 37° C.)through the left ventricle. Retinas were carefully dissected fromenucleated eyes, placed in pre-weighed Eppendorf tubes, dried in speedvacuum overnight and dry weights recorded. Evans blue was extracted byincubating each retina in 120 μl formamide for 18 hours at 70° C.,centrifuged for 2 hours at 6000 rpm. Absorbance of 60 μl of extract wasmeasured at 620 nm and background absorbance was determined at 740 nm.Net absorbance was calculated by subtracting the background 740 nm fromthat at 620 nm. Measurement of a standard curve of Evans blue informamide was also done and Evans blue leakage expressed as μl/g/h wascalculated as shown below:

[EB (ng/ml)×120 (μl)×1000]/[dry weight retina (mg)×circulation time(h)×plasma EB (ng/ml)×100]

As shown in FIG. 1 a below, intravitreal injection of 100 ng VEGFresulted in a 3.5-fold increase (n=17, P<0.05) in vascular permeability18 hours after injection. Treatment with 25 μg/eye of fluocinoloneacetonide and 42.4 μg/eye of compound (1) inhibited the VEGF-inducedleakage by 86.1% and 77.1%, respectively (FIGS. 1 a and 1 b, n=9,P<0.05). Similarly a 10 μg/eye dose of triamcinolone acetonide and itsequivalent dose of compound (18) caused reduction by 67.5% and 54.3%,respectively (FIGS. 2 a and 2 b). In all cases, the inhibition ofleakage was similar in magnitude for both compounds of the invention(compounds (1) and (18)) and its corresponding steroid core.

EXAMPLE F3

Evaluation of the amelioration of steroid-induced intraocular pressure(IOP) elevation in vivo in rats by the compound of the invention.

Test Compounds:

Compound (1) disclosed in Example 1

Fluocinolone acetonide (FC): reference compound

Des-nitro analog of compound (1): Butyl4-((2-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-16,17-16,17-(1-methylethylidenebis(oxy))-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyloxy)-3-methoxybenzoate.

Male Brown Norway rats (250-275 g; Charles River laboratory) wereacclimated for a week before IOP measurements. Baseline IOP measurementswith Tonolab Tonometer (Tiolat Inc) were done in conscious rats (Pease,M. E., et al J. Glaucoma, 15:512-519, 2006) before intravitrealinjection of test compounds. Then the rats were anesthetized withisoflurane inhalation and a drop of 0.5% tetracaine was topicallyapplied on the eyes. Pupils were dilated with 1% topical cyclopentolatehydrochloride and 2.5% phenylephrine HCl to see the needle to aim theintravitreal injection of test compounds. The compound (1) was comparedto corresponding steroid core and to the des-nitro analog at equimolardoses. For example, to compare to fluocinolone acetonide at both 25 and50 μg/eye, compound (1) was dosed at 42.4 and 84.7 μg/eye, respectively.The des-nitro analog of compound (1) was dosed at 39.1 μg/eye(equivalent for 25 μg FC). Control animals received vehicle.

Five measurements were averaged for each time point. IOP measurementswere taken at one and two weeks after intravitreal injection of testcompounds.

Baseline IOP in the Brown Norway rats was 18 mm Hg. As shown in FIG. 3below, one week after injection of fluocinolone acetonide, IOP increasedby 4 mm Hg with 25 μg/eye and 3 mm Hg with 50 μg/eye, respectively. Thiswas maintained after two weeks (p<0.05). However, injection of compound(1) at 25 or 50 μg/eye equivalent (42.4 and 84.7 μg/eye, respectively)caused no change in IOP at one or two week time points (FIG. 3). Inanother experiment seen in FIG. 4 below, des-nitro analog of compound(1) at 25 μg/eye equivalent (39.1 μg/eye) and fluocinolone acetonide(FC) at 25 μg/eye caused increase in IOP compared to compound (1) at 25μg/eye equivalent (42.4 μg/eye).

EXAMPLE F4

Evaluation of the efficacy of the compounds on intraocular pressure,ocular haemodynamics and on retina protection and evaluation ofinflammatory cytokines content in aqueous humor in an in vivoendothelin-1 (ET-1) induced ischemia in New Zealand White rabbits.

Test Compounds

Compound (1) of Example 1

Fluocinolone acetonide (FC): reference compound

Test System and Methods

Twenty adult male New Zealand Albino rabbits weighing 2-2.5 Kg were usedfor the experiments. The animals were divided in two groups for thespecific treatment chosen. The experimental procedures were conform tothose of the Association for Research in Vision and OphthalmologyResolution on the use of animals and in agreement with the GoodLaboratory Practice for the use of animals and were conducted uponauthorization of Italian regulation on protection of animals used forexperimental and other scientific purpose (DM 116/1992) as well as withthe European Union Regulations (OJ of ECL 358/1, Dec. 12, 1986). Therabbits were kept in individual cages, food and water was provided adlibitum. The animals were maintained on a 12-12 h light/dark cycle in atemperature controlled room (22°-23° C.).

Ischemia model of optic injury was obtained throughout by injection,twice a week, for 6 weeks, of endothelin-1 (ET-1) 250 nM, 500 μl ofsterile saline, into the posterior vitreous body of both eyes, using alachrymal cannula, under general anesthesia produced by tiletamine pluszolazepam (Zoletil 100, 0.05 mg/kg) plus xilazine (Xilor 2%, 0.05 ml/kg)i.m.

Fluocinolone acetonide (FC)(0.5 mg/eye in 100 μl of vehicle) or Compound(1) (0.5 mg equivalent/eye in 100 μl of vehicle) were instilledintravitreally (IVT) two weeks after the start of ET-1 treatment (T2),in one eyes, the same volume of vehicle was instilled in the other eye.

Intraocular Pressure

Intraocular pressure (IOP) was measured twice a using a Tono-Pen XL(Medtronic Solan. USA) as described by Maren's group (Exp. Eye Res.(1992) 55: 73-79; Exp. Eye Res. (1993) 57: 67-78) with a two-pointstandard pressure measurement. Two independent investigators (C.U. andR.M.), using the same tonometer performed IOP measurement.

The data reported in Table 2 show that ET-1 treatment did not modify theIOP in New Zealand White rabbits. Fluocinolone acetonide increasedintraocular pressure following ET-1 induced ischemia, on the contraryintraocular pressure following ET-1 induced ischemia was not modifiedwith Compound (1).

TABLE 2 Effect of Fluocinolone acetonide (FC) or compound (1) vs vehicleon IOP. IOP (mmHG) Vehicle FC* Vehicle Compound (1) basal 13.00 ± 2.8313.50 ± 0.17 12.60 ± 2.01 12.70 ± 1.89 ET-1 13.50 ± 0.71 13.00 ± 2.8312.80 ± 1.55 13.30 ± 1.49 I 14.00 ± 2.83 15.50 ± 0.71 13.30 ± 1.49 15.30± 0.95 week II 14.40 ± 0.71 19.00 ± 0.00 13.60 ± 1.96 15.00 ± 1.33 weekIII 15.00 ± 1.41 19.00 ± 1.41 13.40 ± 1.51 13.50 ± 1.08 week IV 13.50 ±1.27 20.80 ± 0.71 14.20 ± 2.15 13.20 ± 1.23 week V 13.30 ± 1.77 22.20 ±0.00 14.10 ± 2.64 15.00 ± 2.45 week VI 13.00 ± 1.56 21.10 ± 0.00 14.80 ±2.90 15.22 ± 1.09 week IOP was taken before daily drug treatment *p <0.001 vs vehicle (N = 10).

Electroretinogram (ERG)

The Electroretinogram (ERG) was performed in basal condition (T0),before the start of drug treatment (T2) and at the end of drug treatment(T6). Slit lamp and indirect funduscopic examinations were performed onall eyes before the study began. Animals demonstrating corneal or lensopacity or retinal damage before the study were excluded. Topicalanaesthesia was applied using one drop 0.2% oxybuprocaine hydrochloride(Novesine, Sandoz). The eyes were dilated by topical application oftropicamide (1%), adapted to darkness for at least 2 hours, and standardERGs recorded in both eyes using corneal electrodes. The reference andground electrodes were made of stainless steel surgical needles, andwere inserted into the ears. The ERG signals were recorded using Retimax(CSO, Florence, Italy). The dark-adapted scotopic response (rodresponse) and scotopic flash response (photopic erg cone), wererecorded. Flashes varied in intensity from −2.50 to +0.4 log scot cds/m². An average of three separate ERGs was determined for each eye. Theamplitude (uV) of a- and b-waves were calculated for each step. Thebaseline values were compared to the response obtained at T2 and at theend of treatment (T6).

ET-1 treatment significantly reduced the amplitude of photopic erg cone(T2, p<0.05 versus T0 and T6 p<0.05 versus T0).

Results reported in Table 3 show that eyes treated with Fluocinoloneacetonide (FC) or Compound (1) exhibited significantly (p<0.05 versusvehicle) less reduction in the ERG wave amplitude than those treatedwith vehicle. Moreover, Compound (1) was slightly more effective thanFluocinolone acetonide.

TABLE 3 Effect of compound (1) or fluocinolone acetonide (FC) vs vehicleon photopic erg cone after ET-1 treatment. Photopic erg cone (amplitude(μV)) Basal Vehicle FC Compound (1) 147.41 ± 7.43 68.85 ± 6.41 136.34 ±11.98 140.94 ± 6.22 * p < 0.001 vs vehicle (N = 8).

Ocular Haemodynamics

Haemodynamic evaluations were performed using an eco-color-dopplerDynaView™ II SSD-1700 (Aloka Holding Europe AG, Milan, Italy). Allanimals underwent Color Doppler Imaging (CDI) investigation before ET-1injection (T0), before the start of drug instillation (T2) and at theend of the drug treatment (T6). Special attention was devoted to theevaluation of ophthalmic and ciliary artery circulation. Blood flowvelocities were measured for each vessel and the Pourcelot ResistanceIndex (RI) was calculated (Galassi F. et al., Acta Opht. Scand Suppl.(2000) 37-38).

Data reported in table 4 show that Fluocinolone acetonide significantlyincreased (p<0.001 versus vehicle) the resistance index in opthalmicartery indicating a decrease in blood perfusion; this effect was notevident with compound (1) treatment.

TABLE 4 Effect of Compound (1) or Fluocinolone acetonide (FC) vs vehicleon ocular haemodinamic evaluated as resistance index Resistence IndexBasal After 2 weeks After 6 weeks Vehicle 0.46 ± 0.07 0.44 ± 0.06 0.48 ±0.05 FC 0.45 ± 0.06 0.45 ± 0.06 0.60 ± 0.04 Compound (1) 0.43 ± 0.060.43 ± 0.08 0.41 ± 0.03 * p < 0.001 vs vehicle (N = 10).

Inflammatory Cytokines in Aqueous Humor

Aqueous humor samples were withdrawn from both anterior and posteriorchamber fluids of each eye before ET-1 administration (T0), before theadministration of Compound (1) or fluorocinolone acetonide (T2), and atthe end of treatments (T6). The same volume of saline were reintroducedevery time. The aqueous humor samples were immediately frozen at −80° C.until use.

Tumor necrosis factor (TNFα) and interleukin 6 (IL-6) were determined inaqueous humor with a commercial kit using an ELISA method (AmershamPharmacia Biotech). The minimum detectable concentrations were 0.10μg/ml for IL-6 and INFα. The interassay coefficient of variation was0.7% for all assays. Data reported in table 5 show that ET-1 treatmentsignificantly increased INFα, IL-6 and VEGF content in aqueous humorsamples and that Compound (1) counteracted these effects moreefficaciously than fluocinolone acetonide (FC).

TABLE 5 Effects Compound (1) and fluocinolone acetonide (FC) on TNFα,IL-6 and VEGF content in aqueous humor. TNFα VEGF IL6 (pg/ml) (pg/ml)(pg/ml) Basal  0.55 ± 0.44 —  0.67 ± 0.43 Vehicle 27.00 ± 4.30 166.76 ±4.54 20.47 ± 2.97 FC 20.16 ± 1.14 114.58 ± 5.61 16.82 ± 0.98 Compound(1) 18.93 ± 0.98 105.79 ± 1.98 15.70 ± 1.73 * p < 0.001 vs vehicle (N =6).

Retina Protection: Morphological Analysis

The eyes of each animal was enucleated, cornea and crystalline wereremoved and the eyes was paraformaldehyde-fixed. Then samples weredehydrated with increasing alcohol concentration (50°-75°-95°-100°).After 95° alcohol treatment, the eyes were divided into two partsfollowing a longitudinal plain from cornea to insertion of opticalnerve. Thus the samples were paraffin-embedded. Section 8 μm thick werestained with Haematoxylin-Eosin. Microscopical fields were registered bydigitizing camera applied a light microscope with a 20× and 40×objective. On the digitized images was performed a morphologicalanalysis of retinal tissue.

The analysis of retinal tissues showed that long term ET-1 treatmentinduced profound morphological changes thereby confirming the functionalimpairment observed in ERG measurements. These morphological changeswere not equally present in animals treated with Compound (1), whereasFluocinolone acetonide was not effective.

DETAILED DESCRIPTION OF DRAWINGS

FIG. 1 a: Measured retinal leakage in VEGF-induced rats: control,treated with compound (1) and its corresponding steroid core,fluocinolone acetonide (FC)

FIG. 1 b: Resultant percent inhibition of leakage by FC and compound(1), derived from FIG. 1 a

FIG. 2 a: Measured retinal leakage in VEGF-induced rats: control,treated with compound (18) and its corresponding steroid core,triamcinolone acetonide (TAAC)

FIG. 2 b: Resultant percent inhibition of leakage by compound (18) andits corresponding steroid core, triamcinolone acetonide (TAAC), derivedfrom FIG. 2 a

FIG. 3: In vivo IOP effects of intravitreally administered fluocinoloneacetonide versus compound (1) in Brown Norway rats

FIG. 4: in vivo IOP effects of intravitreally administered fluocinoloneacetonide (FC), compound (1), and des-nitro analog of compound (1) inBrown Norway rats

1. A compound of formula (I) or a salt or a stereoisomer thereof

wherein R₁ is OH, R₂ is CH₃, or R₁ and R₂ taken together are the groupof formula (II)

R₃ is a hydrogen atom or F and R₄ is F or Cl, with the proviso that:when R₁ is OH and R₂ is CH₃, R₃ is a hydrogen atom; when R₁ and R2 takentogether are the group of formula (II), R₄ is F; R₂, R₃ and R₄ arelinked to the carbon atoms in 17, 16, 6 and 9 of the steroidal structurein position α or β; R is:

wherein Y is selected from: 1) —R₅—CH(ONO₂)R₆ 2)—R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉ 3)—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉ 4)—[(CH₂)_(o)—(X)]_(p)(CH₂)_(q)—CH(ONO₂)—(CR₇R₅)_(n)—(ONO₂)R₉ wherein R₅is a straight or branched C₁-C₁₀ alkylene; R₆ is H or a straight orbranched C₁-C₆ alkyl; R₇ and R₈ at each occurrence are independently Hor a straight or branched C₁-C₆ alkyl; R₉ is H or a straight or branchedC₁-C₆ alkyl; n is an integer from 0 to 6; o is an integer from 1 to 6; pis an integer from 1 to 6; q is an integer from 0 to 6; X is O, S orNR₁₀ wherein R₁₀ is H or a C₁-C₄ alkyl; preferably X is O; excluding thecompounds of formula (I) wherein R₁ and R₂ taken together are the groupof formula (II)

R₄ is F and R₃ is a hydrogen atom, and R is the compound of formula(III) wherein and Y is —R₅—CH(ONO₂)R₆ and R₆ is H.
 2. A compoundaccording to claim 1 wherein R₄ is F, R₃ is F, R₁ and R₂ taken togetherare the group of formula (II) RI, R₂, R₃ and R₄ are linked to the carbonatoms in 17, 16, 6 and 9 of the steroidal structure in position α.
 3. Acompound according to claim 2 wherein Y is 1) —R₅—CH(ONO₂)R₆ wherein R₅is a straight C₁-C₅ alkylene and R₆ is H or —CH₃; or 2)—R₅—CH(ONO₂)—(CR₇R₈)₀—CH(ONO₂)R₉ wherein R₅ is a straight C₁-C₆alkylene, R₉ is H, R₇ and R₈ at each occurrence are independently H orCH₃ and n is 0 or 1; or 3) —[(CH₂)o—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉ whereinRg is H, o is an integer from 2 to 4, p is an integer from 1 to 4, q isfrom 0 to 4 and X is 0;
 4. A compound according to claim 1 wherein R₁and R₂ taken together are the group of formula (II), R₄ is F and R₃ is ahydrogen atom and R₁, R₂, R₃ and R₄ are linked to the carbon atoms in17, 16, 6 and 9 of the steroidal structure in position α.
 5. A compoundaccording to claim 4 wherein Y is: 1) —R₅—CH(ONO₂)R₆ wherein R₅ is astraight C₁-C₆ alkylene and R₆ is —CH₃; or 2)—R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉ wherein R₅ is a straight C₁-C₆alkylene, R₉ is H, R₇ and R₈ at each occurrence are independently H orCH₃, n is 0 or 1; or 3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉ whereinR₉ is H, o is an integer from 2 to 4, p is an integer from 1 to 4, q isfrom 0 to 4 and X is
 0. 6. A compound according to claim 1 wherein R₁ isOH, R₂ is CH₃, R₃ is a hydrogen atom, R₄ is F, and R₁ and R₄ are linkedto the carbon atoms 17 and 9 of the steroidal structure in position α,R₂ is linked to the carbon atom 16 of the steroidal structure inposition β.
 7. A compound according to claim 6 wherein Y is: 1)—R₅—CH(ONO₂)R₆ wherein R₅ is a straight C₁-C₆ alkylene and R₆ is H or—CH₃; or 2) —R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉ wherein R₅ is a straightC₁-C₆ alkylene and R₉ is H; R₇ and R₈ at each occurrence areindependently H or CH₃; n is 0 or 1; or 3)—[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉ wherein R₉ is H, o is an integerfrom 2 to 4, p is an integer from 1 to 4, q is from 0 to 4 and X is O,8. A compound according to claim 1 wherein R₁ is OH, R₂ is CH₃, R₃ is ahydrogen atom and R₄ is Cl; R₁ and R₄ are linked to the carbon atoms 17and 9 of the steroidal structure in position α, R₂ is linked to thecarbon atom 16 of the steroidal structure in position β.
 9. A compoundaccording to claim 8 wherein Y is 1) —R₅—CH(ONO₂)R₆ wherein R₅ is astraight C₁-C₆ alkylene and R₆ is H or —CH₃; or 2)—R₅—CH(ONO₂)—(CR₇R₈)_(n)—CH(ONO₂)R₉ wherein R₅ is a straight C₁-C₆alkylene and R₉ is H; R₇ and R₈ at each occurrence are independently Hor CH₃; n is 0 or 1; or 3) —[(CH₂)_(o)—X]_(p)—(CH₂)_(q)—CH(ONO₂)R₉wherein R₉ is H, o is an integer from 2 to 4, p is an integer from 1 to4, q is from 0 to 4 and X is 0;
 10. A compound according to claim 1selected from:


11. A compound according to claim 1 for use as medicament.
 12. Acompound according to claim 1 for use in the treatment of inflammatorydiseases.
 13. A compound according to claim 1 for use in the treatmentof ocular diseases.
 14. A compound according to claim 13 wherein oculardiseases include diabetic macular edema, diabetic retinopathy, maculardegeneration, age-related macular degeneration and other diseases ofretina and macula lutea.
 15. Use of a compound according to claim 1 forthe preparation of a medicament for the treatment of inflammatorydiseases.
 16. Use of a compound according to claim 1 for the preparationof a medicament for the treatment of ocular diseases.
 17. Pharmaceuticalcomposition comprising a pharmaceutically effective amount of at least acompound according to claim 1 and ophthalmically acceptable excipientsin a suitable form for intravitreal or periorbital administration. 18.Pharmaceutical composition according to claim 17 for use in thetreatment of inflammatory diseases.
 19. Pharmaceutical compositionaccording to claim 17 for use in the treatment of ocular diseases.
 20. Acompound of formula (I) or a salt or a stereoisomer thereof

for use in the treatment of ocular diseases, wherein in formula (I) R₁and R₂ taken together are the group of formula (II)

R₃ is a hydrogen atom, R₄ is F, R₁, R₂ and R₄ are linked to the carbonatoms in 17, 16 and 9 of the steroidal structure in position α; R is:

Y is —R₅—CH(ONO₂)R₆ and R₆ is H.
 21. A compound according to claim 20wherein ocular diseases include diabetic macular edema, diabeticretinopathy, macular degeneration, age-related macular degeneration andother diseases of retina and macula lutea.
 22. The compound according toclaim 20 of formula (18)


23. Pharmaceutical composition comprising a pharmaceutically effectiveamount of at least a compound of claim 20 and ophthalmically acceptableexcipients in a suitable form for intravitreal or periorbitaladministration.
 24. Pharmaceutical composition according to claim 23 foruse in the treatment of ocular diseases.